title: Recombinant carboxyl-terminal fibrin-binding domain of human fibronectin expressed in mouse L cells.
abstract: The carboxyl-terminal fibrin-binding domain, Fib2, of human fibronectin was expressed in mouse L cells as a fusion protein with the signal sequence of human protein C inhibitor. The recombinant Fib2 (rFib2) protein synthesized by transfected cells retained the ability to form dimers with each other or with mouse fibronectin subunits and was secreted to the medium after extensive glycosylation. Only a small fraction of the secreted protein was incorporated into the pericellular matrix. Interestingly, the secreted rFib2 protein displayed a remarkable heterogeneity upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, giving rise to a broad band corresponding to Mr of 60,000-90,000. The heterogeneity was eliminated mostly by treatment with neuraminidase and further by treatment with endo-alpha-N-acetylgalactosaminidase. Treatment with peptide:N-glycosidase F did not alter the heterogeneity of the protein, indicating that differential sialylation of O-linked, but not N-linked, glycans is largely responsible for the apparent heterogeneity. The presence of O-linked but absence of N-linked glycans was further supported by the observations that peanut agglutinin specifically bound to the desialylated rFib2 protein, whereas neither concanavalin A nor lentil lectin bound to the protein irrespective of prior neuraminidase treatment. Since the apparent heterogeneity of the rFib2 protein was only observable with the secreted, but not the cytoplasmic form, sialylation of O-linked glycans may be essential for, or regulate as a rate-limiting step, the transit of the recombinant protein to the extracellular space.
authors.affiliation: Institute for Comprehensive Medical Science, Fujita-Gakuen Health University School of Medicine, Aichi, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Ichihara-Tanaka
authors.forename: K
authors.initials: K
authors.lastname: Titani
authors.forename: K
authors.initials: K
authors.lastname: Sekiguchi
mesh_terms: D000818:Animals; D001665:Binding Sites; D004591:Electrophoresis, Polyacrylamide Gel; D005337:Fibrin / Q000378:metabolism*; D005353:Fibronectins / Q000235:genetics / Q000378:metabolism*; D006596:Hexosaminidases / Q000494:pharmacology; D006801:Humans; D007739:L Cells; D046911:Macromolecular Substances; D051379:Mice; D008970:Molecular Weight; D009439:Neuraminidase / Q000494:pharmacology; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D014162:Transfection; D048809:alpha-N-Acetylgalactosaminidase
chemical_list: D005353:Fibronectins; D046911:Macromolecular Substances; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D005337:Fibrin; D006596:Hexosaminidases; D009439:Neuraminidase; C482722:NAGA protein, human; D048809:alpha-N-Acetylgalactosaminidase